Method for testing the differentiation status in pancreatic cells of a mammal

ABSTRACT

The present invention relates to a novel method for testing the developmental status in pancreatic cells of a mammal. The present invention further relates to applications in the medical field that directly arise from the method of the invention. Additionally, the present invention relates to transgenic mammals comprising at least one inactivated Pax4 allele and optionally at least one inactivated Pax6 allele.

RELATED APPLICATIONS

This application is a division of application Ser. No. 08/778,394 filedon Dec. 31. 1996, which is a continuation-in-part of U.S. applicationSer. No. 08/381,841, filed Mar. 27, 1995 now U.S. Pat. No. 5,747,250, asthe National Phase of PCT/EP93/02051, filed Aug. 2, 1993, designatingthe U.S. and claiming priority from German application P 42 25 569.4,filed Aug. 3, 1992. Each of the aforementioned U.S., PCT, and Germanapplications are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for testing the differentiationstatus of pancreatic cells in a mammal; for instance, by ascertainingthe level or status of Pax4 mRNA and/or protein in pancreatic cells (orpre-pancreatic cells) and comparing the level or status with thecorresponding level or status in normal pancreatic (or pre-pancreatic)cells. This method provides a means for diagnosis or detection ofdiseases which arise from certain pancreatic cells, especially a meansfor diagnosis or detection of, for instance, diabetes, such as juvenilediabetes. The method can be performed in conjunction with ascertainingthe level or status of Pax6 mRNA and/or protein in pancreatic cells (orpre-pancreatic cells) and comparing the level or status with thecorresponding level or status in normal pancreatic (or pre-pancreatic)cells.

The data set forth below shows that deficiency in Pax4 expression isindicative of deficiency or failure in β-cell development and ergoinsulin production (and thus diabetes such as juvenile diabetes). Theinvention thus relates to restoring Pax4 expression for treatment,prevention or delaying a pancreatic disease such as diabetes, e.g.,juvenile diabetes; and ergo transgenic mammals having restored Pax4expression by modification so as to comprise at least one first nucleicacid molecule having a sequence encoding a functional and expressiblePax4 protein and optionally a second nucleic acid sequence encoding afunctional and expressible Pax6 protein. Alternatively or additionally,the invention relates to administration of Pax4 alone or with Pax6and/or of an agent for stimulating expression of Pax4 or Pax4 and Pax6,for treatment, prevention or delaying a pancreatic disease such asdiabetes, e.g., juvenile diabetes.

Since the data set forth below shows that deficiency in Pax4 expressionis indicative of deficiency or failure in β-cell development, and ergoinsulin production (and thus diabetes such as juvenile diabetes), thepresent invention also relates to transgenic mammals modified so as tocomprise at least one inactivated Pax4 allele. This mammal has numerousutilities, including as a research model for pancreatic diseases such asjuvenile diabetes; and therefore, presents a novel and valuable animalin the development of therapies, treatment, etc. for diseases caused bydeficiency or failure of pancreatic cells. Accordingly, in thisinstance, the mammal is preferably non-human, e.g., a laboratory animalsuch as a mouse or rat.

Further, since improper expression of Pax4 may also cause maladies suchas tumors, the invention also relates to a transgenic mammal modified soas to comprise at least one inactivated Pax4 allele for treatment,prevention or the delay of a pancreatic disease caused by improperexpression of Pax4, such as tumors. In this instance, the mammal can bea human, as the introduction into the mammal of the at least oneinactivated Pax4 allele is for therapy. Alternatively or additionally,the invention relates to administration of an agent which inhibits Pax4or Pax4 and Pax6 for treatment, prevention or delaying a pancreaticdisease caused by improper expression of Pax4, such as tumors.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including manufacturer'sspecifications, instructions, etc.) are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

The pancreas is an essential organ possessing both an exocrine functioninvolved in the delivery of enzymes into the digestive tract and anendocrine function by which various hormones are secreted into the bloodstream. The exocrine function is assured by acinar and centroacinarcells that produce various digestive enzymes (amylase, proteases,nuclease, etc.) and intercalated duct cells that transport these enzymesin alkaline solution to the duodenum.

The functional unit of the endocrine pancreas is the islet of Langerhanswhich are scattered throughout the exocrine portion of the pancreas andare composed of four cell types: α-, β-, δ- and PP-cells, reviewed inSlack, Development 121 (1995), 1569-1580. β-cells produce insulin,represent the majority of the endocrine cells and form the core of theislets, while α-cells secrete glucagon and are located in the periphery.δ-cells and PP-cells are less numerous and respectively secretesomatostatin and a pancreatic polypeptide. Insulin and glucagon are keyregulators of blood glucose levels. Insulin lowers blood glucose levelby increasing the cellular uptake of glucose and its conversion toglycogen. Glucagon elevates blood glucose levels by intervening with thebreakdown of liver glycogen. Common pancreatic disorders affectingendocrine function include diabetes mellitus and hormone secretingtumors.

All four endocrine cells are thought to originate from a commonpluripotent precursor that is derived from the endoderm. Early duringpancreatic development, these precursors co-express several hormonessuch as insulin and glucagon. In mouse, the α-cells are the firstendocrine cells to differentiate at day 9.5 post-conception (p.c.),followed by the β- and δ-cells at day 14.5 p.c. and the PP-cells atpostnatal day 1. Very little is known on the molecular and geneticfactors involved in defining the lineage of the different endocrinecells. One of the few genes described so far is the homeobox gene Pdx1which is expressed during the initial stages of pancreatic developmentand becomes restricted to the β-cells in adult islets (Guz et al.,Development 121 (1995), 11-18). Homozygous mouse Pdx1 mutants fail todevelop a pancreas and die a few days after birth (Jonsson et al.,Nature 371 (1994), 606-609). Two members of the Pax gene family, Pax4and Pax6, are also expressed in endocrine cells during pancreaticdevelopment. Until now, however, it was not known how in particular thePax4 and the Pax6 gene affects pancreatic development.

A method for testing for a variety of differentiation parameters in thepancreas was hitherto not available but is nevertheless highlydesirable. Results obtainable by such a method have a significant impacton, e.g., the diagnosis and therapy of pancreas related diseases.

SUMMARY OF THE INVENTION

The present invention relates to a novel method for testing thedifferentiation status of pancreatic cells in mammals. The presentinvention further relates to applications in the medical field thatdirectly arise from the method of the invention. Additionally, thepresent invention relates to transgenic mammals comprising at least oneinactivated Pax4 gene and optionally at least one inactivated Pax6 gene.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, a technical problem underlying the present invention was toprovide such a method for monitoring the differentiation status ofpancreatic cells. The solution to said technical problem is achieved bythe embodiments characterized in the claims.

Thus, the present invention relates to a method for testing thedifferentiation status in pancreatic cells of a mammal comprising

(a) determining the level or status of Pax4 mRNA in pancreatic cells ofsaid mammal; and/or

(b) determining the level or status of Pax4 protein in pancreatic cellsof said mammal; and

(c) comparing said level or status of Pax4 mRNA and/or Pax4 protein withthe corresponding level in normal pancreatic cells.

In connection with the present invention, the term "level" denotes theamount of mRNA or protein produced. The term "status" includes theoptions that the gene, mRNA, protein or a transcription control element,e.g. promoter/enhancer sequence may bear a mutation, deletion or anyother modifications which would affect the overall activity of the genewhen compared to the wild-type normal gene product. Included in thisterm are post-translational modifications of the protein.

The method of the present invention allows for the first time a detailedstudy of the development of different cell types, i.e. α-, β-, δ- andPP-cells in the pancreatic tissue. As is demonstrated by the appendedexamples, the Pax4 gene, optionally in conjunction with the Pax6 gene isinvolved in the early steps of pancreatic development. This surprisingresult allows the monitoring of cell fate as well as the investigationof the development of diseases which arise from certain cell typescontained in the pancreas. The results presented in accordance with thepresent invention furthermore allow the conclusion that Pax4 and/or Pax6are master regulators for specific pancreatic cells. Thus, Pax4 appearsto be a master regulator of β-cells. Pax4 is also expressed innon-pancreatic cells. Pax4 expression is detected in a subset of cellsfound in the developing spinal cord. Therefore, Pax4 may likewise play arole in the differentiation status of these cells.

Accordingly, the method of the present invention provides a means fordiagnosis or detection of diseases which arise from certain pancreaticcells, especially a means for early detection or diagnosis; forinstance, detection or diagnosis of diabetes, such as juvenile diabetes;and, such detection or diagnosis can be pre- or post-natal.

In a preferred embodiment, the method of the present invention furthercomprises

(d) determining the level or status of Pax6 mRNA in pancreatic cells ofsaid mammal; and/or

(e) determining the level or status of Pax6 protein in pancreatic cellsof said mammal; and

(f) comparing said level or status of Pax6 mRNA and/or Pax6 protein withthe corresponding level in normal pancreatic cells.

This embodiment of the present invention allows the study of thesynergistic effects of the Pax4 and the Pax6 gene products. It isexpected that the analysis of said synergistic effect provides deeperinsights into the regulation of cell specific development in thepancreas. From said deeper insight the development of diagnostic andpharmaceutical compositions related to pancreas-specific diseases willgreatly benefit.

In a further preferred embodiment of the method of the invention, saidmammal is in the

(i) embryonic;

(ii) newborn; or

(iii) adult stage.

As has been shown in accordance with the present invention, the Pax4gene, preferably in conjunction with the Pax6 gene, is expressed at adifferent level and at different stages of mammalian pancreasdevelopment. The specific analysis of the developmental stage ofpancreatic cells at the embryonic, newborn or adult stage will providefurther insights into, e.g., specific disease states associated with therespective stages. For example, it is expected that the etiology of,e.g., juvenile diabetes will be elucidated by applying the method of thepresent invention, as well as by employing the transgenic mammals(non-human) according to the invention (discussed infra; see also theexamples). Upon the basis of this knowledge, new pharmaceutical activedrugs, e.g. against juvenile diabetes, will be developed and tested.

The method of the invention can be applied to a variety of mammals,depending on the purpose of the investigation. Thus, in a preferredembodiment, the mammal is a mouse. This embodiment is particularlyuseful for basic research to understand more clearly the functionalinterrelationship of different proteins which regulate the developmentof the pancreas. In a further embodiment the mammal is a human. In thisembodiment, preferably diagnostic and therapeutic applications areenvisaged.

In a further preferred embodiment of the method of the invention, steps(a) and optionally (d) and/or (b) and optionally (e) are carried out invivo.

This embodiment of the invention is expected to be useful in particularin basic research or in therapeutic applications.

In a further preferred embodiment of the method of the invention, steps(a) and optionally (d) and/or (b) and optionally (e) are carried out invitro.

The advantages of this embodiment would be expected to lie primarily indiagnostic applications and, again, in basic research.

In a further preferred embodiment of the method of the invention, saiddetermination in step (a) and optionally in step (d) is effected byemploying

(i) a nucleic acid sequence corresponding to at least a part of the Pax4gene and preferably encoding at least part of the Pax4 protein andoptionally a second nucleic acid sequence corresponding to at least apart of the Pax6 gene and preferably encoding at least part of the Pax6protein;

(ii) a nucleic acid sequence complementary to the nucleic acidsequence(s) of (i); or

(iii) a primer or a primer pair hybridizing to the nucleic acidsequence(s) of (i) or (ii).

In accordance with this embodiment of the present invention, the methodof testing the differentiation status can be effected by using a nucleicacid molecule encoding the Pax4 gene and/or the Pax6 gene or a partthereof, e.g., in the form of a Southern or Northern blot or in situanalysis. Said nucleic acid sequence may hybridize to a coding region ofeither of the genes or to a non-coding region. In the case that acomplementary sequence in accordance with (ii) is employed in the methodof the invention, said nucleic acid molecule can again be used inNorthern blots. Additionally, said testing can be done in conjunctionwith an actual blocking, e.g., of the transcription of the gene and thusis expected to have therapeutic relevance. Furthermore, a primer oroligonucleotide can also be used for hybridizing to one of theabove-mentioned Pax genes or corresponding mRNAs. The nucleic acids usedfor hybridization can, of course, be conveniently labeled byincorporating or attaching, e.g., a radioactive or other marker. Suchmarkers are well known in the art. The labeling of said nucleic acidmolecules can be effected by conventional methods.

Additionally, the presence or expression of Pax4 and optionally Pax6 canbe monitored by using a primer pair that specifically hybridizes toeither of the corresponding nucleic acid sequences and by carrying out aPCR reaction according to standard procedures.

Specific hybridization of the above mentioned probes or primerspreferably occurs at stringent hybridization conditions. The term"stringent hybridization conditions" is well known in the art; see, forexample, Sambrook et al., "Molecular Cloning, A Laboratory Manual"second ed., CSH Press, Cold Spring Harbor, 1989; "Nucleic AcidHybridisation, A Practical Approach", Hames and Higgins eds., IRL Press,Oxford, 1985.

Further modifications of the above-mentioned embodiment of the inventioncan be easily devised by the person skilled in the art, without anyundue experimentation from this disclosure.

An additional embodiment of the present invention relates to a methodwherein said determination in step (b) and optionally of step (e) iseffected by employing an antibody or fragment thereof that specificallybinds to the Pax4 protein and optionally by employing a second antibodyor fragment thereof which specifically binds to the Pax6 protein.

Antibodies or fragments thereof to the aforementioned protein can beobtained by using conventional methods which are described, e.g., inHarlow and Lane "Antibodies, A Laboratory Manual", CSH Press, ColdSpring Harbor, 1988. These antibodies may be monoclonal antibodies orcomprised in polyclonal antisera or fragments thereof. The antibody usedin the method of the invention may be labeled with detectable tags suchas a histidine flags or a biotin molecule.

In a further preferred embodiment of the method of the presentinvention, said pancreatic cells are β-cells or δ-cells.

In accordance with the present invention, it was found that Pax4 isexpressed in β-cells. Disruption of the Pax4 gene function thereforeallows a close monitoring of the development of said cells in thepancreas. Since Pax6 is also expressed in both α- and β-cells (seeconcurrently filed application Ser. No. 08/778,394 (attorney docket674503-2002, incorporated herein by reference), Pax6 expression may berequired to establish a competent background for Pax4 to act.

Since both Pax4 and Pax6 are regulatory proteins required for properdifferentiation of endocrine cells, this embodiment may allow a varietyof conclusions with regard to the generation of diseases such aspancreatic tumors.

Accordingly, a further preferred embodiment relates to a method whereinsaid differentiation status is indicative of a malignancy or malignantpotential of said pancreatic cell. Overexpression or absence of afunctional Pax4 and optionally Pax6 product may induce normal endocrinecells to become cancerous. In accordance with this statement, allelicdeletions in chromosome 7 q in the vicinity of the Pax4 gene areobserved in many pancreatic carcinomas (Alberto et al., Cancer Res. 56(1996), 3808-3818). Pax4, and optionally Pax6, may also interact withother oncogenic factors.

Malignancy or malignant potential is used in accordance with thisinvention preferably but not exclusively in connection with pancreatictumors such as insulinoma, glucagonomas, somatostatinomas and ductalcell adenocarcinomas.

The present invention relates in a further preferred embodiment to amethod that further comprises

(a') prior to said testing removal of a solid pancreatic tumor from saidmammal; and

(b') after said testing, at least partial elimination of the expressionof the Pax4 and optionally the Pax6 gene in cells of said tumor, if saidgene(s) is/are over-expressed or stimulation of expression of the Pax4gene and optionally the Pax6 gene or introduction of a functional andexpressible Pax4 gene and optionally a functional and expressible Pax6gene into said cells if said gene(s) is/are under-expressed or notexpressed; and

(b") reintroducing the cells obtained as a product of step (b') intosaid mammal.

In this context and as used throughout this specification, "functional"Pax4 (Pax6) means a protein having part or all of the primary structuralconformation of the Pax4 (Pax6) protein possessing the biologicalproperty of contributing to the development of endocrine cells intoβ-cells (α-cells and Langerhans cells), said protein being the productof procaryotic or eukaryotic expression of a Pax4 (Pax6) encoding DNAsequence and having an amino acid sequence comprising the amino acidsequence of SEQ ID No. 2 (SEQ ID No. 4) or any fragment or derivativethereof by way of amino acid deletion, substitution, insertion, additionand/or replacement of the amino acid sequence given in SEQ ID No. 2 (SEQID No. 4). Also comprised by the term "functional" Pax4 (Pax6) proteinis the capability of said protein or part thereof to generate a specificimmune response such as an antibody response.

This embodiment of the present invention is suited for therapy oftumors, in particular in humans. Therefore, it is envisaged thatpancreatic tumor cells are monitored for the expression level of thePax4 protein and optionally Pax6 protein. Detection of anover-expression of said protein(s) would allow the conclusion that saidover-expression is interrelated to the generation or maintenance of thetumor. Accordingly, a step would be applied to reduce the expressionlevel to normal levels. This can be done, for example, by at leastpartial elimination of the expression of the Pax4 gene by biologicalmeans, for example, by the use of ribozymes, antisense nucleic acidmolecules or intracellular antibodies against either the Pax4 or Pax6protein. Furthermore, pharmaceutical products may be developed thatreduce the expression levels of Pax4. While it is presently unclear howPax4 and optionally Pax6 are regulated in pancreatic tissue, it ispossible that different developmental and hormonal factors determine thelevels of activity of these genes. For example, small molecules areknown to repress the expression of certain genes. It has beendemonstrated that activin A, a member of the TGFβ superfamily, candownregulate Pax6 expression in the developing spinal cord (Pituello etal., Proc. Natl. Acad. Sci. USA 92 (1995), 6952-6956). Similar moleculesmay downregulate Pax4 or optionally Pax6 expression in the pancreas. Onthe other hand, lack of expression or under-expression may be remediedby a functional Pax4 gene and optionally a functional Pax6 gene whichshould both be expressible in the tumor cells. Stimulation or inductionof expression can be obtained again by the use of small molecules orother means, this time activating Pax4 gene expression. In this regard,it is important to note that the present invention envisages thepossibilities that one of said Pax genes is over-expressed whereas thesecond Pax gene is under-expressed in said malignant state. Finally,surgical removal or chemotherapeutic treatment of pancreatic tumors inhumans often leaves the patient without a significant number of insulinproducing cells. Pax4 and optionally Pax6 may be used in tissueengineering (Langer and Vacanti, Science 260 (1993), 920-924) for thedevelopment of functional substitute for missing or damaged β-cells.Pancreatic tumor cells that have reverted in vitro to a normal levels ofPax4 expression, and optionally of Pax6 expression, can be re-introducedinto the patient so that the said patient is provided with normalinsulin producing cells of his own genetic background thereby reducingthe risk of immunological rejection of the cells.

In a further preferred embodiment of the present invention, the testingfor differentiation status in pancreatic cells is a testing for thedevelopmental status in β-cells, which as shown by the examples, isindicative of juvenile diabetes. Juvenile diabetes is often the resultof deficiency or failure in β-cell development. The examples show thatdeficiency in Pax4-expression is indicative of deficiency or failure inβ-cell development and ergo insulin production (and thus diabetes suchas juvenile diabetes).

Early diagnosis of juvenile diabetes is particularly advantageous and ofconsiderable medical importance. Thus, it is a preferred embodiment toemploy methods of the invention for diagnosis or detection of diabetes.This preferred embodiment can be used to diagnose juvenile diabetes inthe coronar villi, i.e. prior to the implantation of the embryo.Furthermore, juvenile diabetes can, with the method of the presentinvention, be diagnosed via amniocentesis. The early diagnosis ofjuvenile diabetes in accordance with all applications of the method ofthe invention allows for treatment directly after birth before the onsetof clinical symptoms.

In a particularly preferred embodiment of the method of the invention,said testing for differentiation status in said β-cells is carried outin an embryonic or newborn mammal.

As has been indicated hereinabove, it is particularly preferred toinclude at least one further step in the method of the invention, whichis specific for different pharmaceutical and genetic therapeuticapproaches. As mentioned above, different small pharmaceutically activemolecules could be used to activate Pax4 and optionally Pax6 expressionand therefore induce differentiation and production of insulin producingβ-cells. Likewise, intracellular targeting of active Pax4 and/oroptionally Pax6 would advance similar consequences. In accordance withthis statement, pancreatic ductal epithelial cells have been proposed tocontain potential stem cells for endocrine cell types. Induction of Pax4activity in said cells but not exclusively in said cells can promotedifferentiation into insulin producing cells (Parsa et al, 1985, CancerRes. 45:1285-1290).

In a further preferred embodiment of the method of the invention, saiddifferentiation status of pancreatic cells is the result of the activityof a medicament or of a gene therapy approach. For example, saiddifferentiation status may be influenced by gene therapy approacheswhere a functional Pax4 and optionally Pax6 gene is introduced in vivointo cells using a retroviral vector (Naldini et al., Science 272(1996), 263-267; Mulligan, Science 260 (1993), 926-932) or anotherappropriate vector. Likewise, in accordance with the present inventioncells from a patient can be isolated, modified in vitro to differentiateinto β-cells using standard tissue culture techniques and reintroducedinto the patient.

In a particularly preferred embodiment, said medicament or gene therapyapproach affects the expression level of the Pax4 gene and, optionally,of the Pax6 gene at the mRNA or protein level.

The above embodiments of the present invention allow, inter alia,testing of a medicament for its influence on expression of theaforementioned Pax genes. As has been stated further hereinabove,abnormal expression levels of Pax4 and optionally of Pax6 are expectedto be a causative agent in the generation of, for example, solid tumorsof the pancreas. The method of the invention thus allows the testing ofmedicaments, the application of which would allow the cell to return toa normal expression level. Said normal expression level would beexpected to have a direct influence on, e.g., the malignancy of a cell.For example, if a disease or tumor is a direct or indirect result of anunder-expression of Pax4, the physician treating the respective patientwould administer a medicament that stimulates expression of Pax4.

In a further preferred embodiment of the method the invention, thetesting for differentiation status in pancreatic cells is a testing forthe developmental status in Pax4 knockout mice that are optionally atthe same time Pax6 knockout mice.

In an additional preferred embodiment of the method of the presentinvention, said method further comprises introducing a functional andexpressible Pax4 gene and optionally further comprising introducing afunctional and expressible Pax6 gene into pancreatic α-cells or ductalepithelial cells which possess a similar yet different differentiationpathway as compared to β-cells. With this embodiment of the invention,the person skilled in the art is in the position to redirect the fate ofα-cells or ductal epithelial cells into β-cells. Thus, α-cells areexpected to differentiate after transfection with the Pax4 gene andoptionally the Pax6 gene into β-cells. A corresponding pharmaceuticalapplication is envisaged, if a patient suffers from a β-cell- and thusinsulin deficiency. It is envisaged that this method is carried out invitro or in vivo.

The present invention further relates to a transgenic mammal comprisingat least one inactivated Pax4 allele. As to research uses of thetransgenic mammal especially, it is preferred that the mammal benon-human.

The transgenic animal of the present invention can advantageously beused for monitoring the development of different cells, for example, inthe pancreas. However, the use of the transgenic mammal is not confinedto the study of pancreatic development. Since Pax4 is, in accordancewith the present invention, now believed to be a master regulator forβ-cells, its influence can also be studied in other cell types of thebody.

Since the transgenic animal of the invention which is preferably atransgenic mouse in the homozygous state has severe pancreatic disordersthat, in the case of transgenic mice, lead to death within three daysafter birth, said transgenic animal can further be used for theinvestigation of diseases associated with developmental disorders, inparticular in the pancreas. Since the transgenic mice are deficient ininsulin producing cells and present clinical symptoms similar to humanpatients suffering from juvenile diabetes, said mice can serve has ananimal model for therapeutic and pharmaceutical research againstjuvenile diabetes.

Preferably, the transgenic mammal of the invention further comprises atleast one inactivated Pax6 allele.

This embodiment allows the study of the interaction of Pax4 and Pax6 onthe development of the mammal or certain tissues thereof, in particular,of the pancreas. All the applications that have been herein beforediscussed with regard to the Pax4 transgenic mammal also apply to themammal that carries two transgenes. It might be also desirable toinactivate Pax4 gene expression and optionally Pax6 gene expresssion ata certain stage of development and/or life of the transgenic animal.This can be achieved by using, e.g., tissue specific developmentaland/or cell regulated and/or inducible promoters which drive theexpression of, e.g., an antisense or ribozyme directed against the RNAtranscript encoding the Pax4 protein and optionally to the Pax6 encodingRNA. A suitable inducible system is for example thetetracycline-regulated gene expression as described, e.g., by Gossen andBujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen etal. (Trends Biotech. 12 (1994), 58-62).

In another preferred embodiment of the invention said transgenic mammalis human, a mouse or a rat. Since at least one inactivated Pax4 allelecan be introduced into a mammal for therapeutic applications, asdiscussed above, the transgenic mammal can be human.

In accordance with the present invention, the transgenic animal may behomozygous or heterozygous for either inactivated Pax4 or inactivatedPax6 or for both inactivated genes.

Moreover, the present invention relates to the use of at least one firstnucleic acid sequence encoding a functional and expressible Pax4 proteinand optionally a second nucleic acid sequence encoding a functional andexpressible Pax6 protein for the preparation of a pharmaceuticalcomposition for treating, preventing and/or delaying diabetes in amammal. According to the invention, vectors containing said nucleic acidsequences may be operatively linked to regulatory elements allowing forexpression of said nucleic acid sequences and/or for targeting of saidnucleic acid sequences to pancreatic cells.

Further, the invention relates to the use of a functional Pax4 proteinand optionally a functional Pax6 protein for the preparation of apharmaceutical composition for the treatment, prevention and/or delay ofdiabetes and/or a neuronal disorder in a mammal. The term "functional"bears the same meaning as outlined hereinabove.

Preferably, the mammal referred to in the above embodiments is a human,a rat or a mouse. And thus, the invention further comprehends atransgenic mammal modified so as to comprise at least one first nucleicacid molecule comprising a sequence encoding a functional andexpressible Pax4 protein and optionally a second nucleic acid moleculecomprising a sequence encoding a functional and expressible Pax6 protein(wherein the mammal has expression of the nucleic acid molecule(s)). Themammal can be modified pre-natally or post-natally, e.g., after a methodof the present invention shows low, impaired or no Pax4 protein or mRNA,for treatment, prevention or delaying diabetes; and, the modificationcan be by techniques discussed herein or by techniques within the ambitof the skilled artisan, without undue experimentation from thisdisclosure.

It is envisaged by the present invention that the nucleic acids andproteins are administered either alone or in any combination, andoptionally together with a pharmaceutically acceptable carrier orexcipient. Said nucleic acid sequences may be stably integrated into thegenome of the mammal. On the other hand, viral vectors may be used whichare specific for certain cells or tissues, preferably pancreas and/orbrain, and which persist in said cells thereby conferring expression ofthe Pax genes in said cells. Suitable pharmaceutical carriers andexcipients are well known in the art. Elements capable of targeting anucleic acid molecule and/or protein to specific cells are described inthe prior art, for example in, Somia et al., Proc. Natl. Acad. Sci. USA92 (1995), 7570-7574. The pharmaceutical compositions can beadministered to the mammal at a suitable dose, which can be determinedfrom this disclosure and knowledge in the art, without undueexperimentation by the skilled artisan taking into consideration typicalfactors such as the species, age, sex, weight, condition of the mammal,the route of administration, whether a Pax4 protein or Pax4 and Pax6proteins are being administered, whether an agent for inhibiting orstimulating Pax4 or Pax4 and Pax6 is being administered, whether anucleic acid or acids are being administered, and whether the nucleicacid or acids are for expression of Pax4 or Pax4 and Pax6 or are forinhibiting expression of Pax4 or Pax4 and Pax6, inter alia. A typicaldose can be, for example, in the range of 0.001 to 1000 μg (or ofnucleic acid for expression or for inhibition of expression in thisrange); however, doses below or above this exemplary range areenvisioned, especially considering the aforementioned factors.

Administration of the suitable compositions may be effected in differentways, e.g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration. The pharmaceuticalcompositions prepared according to the invention can be used for theprevention or treatment or delaying of different kinds of diseases, forexample, pancreas related diseases, namely diabetes or different kindsof acquired or in-born neural disorders, neural degenerations andrelated disorders. Said diseases and disorders are preferably derivedfrom endocrine or neural tissues, e.g. pancreas and brain.

Furthermore, it is possible to use a pharmaceutical composition whichcomprises a nucleic acid sequence which encodes a Pax4 protein andoptionally a nucleic acid sequence encoding a Pax6 protein for genetherapy. Naturally, both sequences may also be comprised in the samevector. As described above, the diseases often evolve when cells loseboth functional copies of the Pax genes. In such a case, introduction offunctional copies of the corresponding gene may help to ameliorate thesituation. For example, research pertaining to gene transfer into cellsof the germ line is one of the fastest growing fields in reproductivebiology. Gene therapy, which is based on introducing therapeutic genesinto cells by ex-vivo or in-vivo techniques is one of the most importantapplications of gene transfer. In genetic diseases the introduction of anormal or a functionally adequate allele of a mutated nuclear generepresents gene replacement therapy, which is applicable mainly tomonogenetic recessive disorders such as, for example, diabetes andhypoglycemia.

Thus, in a further embodiment, the invention relates to a method fortreating diabetes comprising:

(a) removal of a cell from a mammal;

(b) introduction of a functional and expressible Pax4 gene andoptionally a functional and expressible Pax6 gene into said cell; and

(c) reintroducing the cell obtained as a product of step (b) into saidmammal or into a mammal of the same species.

Yet, in a further embodiment, the invention relates to a method fortreating a neuronal disorder comprising:

(a) removal of a cell from a mammal;

(b) introduction of a functional and expressible Pax4 gene andoptionally a functional and expressible Pax6 gene into said cell; and

(c) reintroducing the cell obtained as a product of step (b) into saidmammal or into a mammal of the same species.

It is to be understood that the introduced genes are functional andexpressible after introduction into said cell and preferably remain inthis status during the lifetime of said cell.

Preferably, said mammal is a human, a rat or a mouse.

In a preferred embodiment of the method of the invention, said cell is agerm line cell or embryonic cell or derived therefrom. In a furtherpreferred embodiment, said cell is an egg cell or derived therefrom.

Suitable vectors and methods for in-vitro or in-vivo gene therapy aredescribed in the literature and are known to the person skilled in theart. The pharmaceutical compositions according to the invention can beused for the treatment of kinds of diseases hitherto unknown as beingrelated to the expression and/or non-expression of the Pax4 gene and/orthe Pax6 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

This disclosure may best be understood in conjunction with theaccompanying drawings, incorporated herein by reference, which show:

FIG. 1 (FIGS. 1a, 1b, 1c, 1d). Targeted disruption of Pax4 andgeneration of Pax4 (-/-) mice

a, structure of the wild-type and targeted Pax4 loci. A targeteddeletion of almost all the entire paired box domain (dark boxes; exons2, 3 and 4) of Pax4 was produced, by fusing in frame aβ-galactosidase-neomycin resistance cassette (transcription direction isindicated by double arrowheads). Restriction enzymes: A, ApaI; K, KpnI;Nh, NheI; N, NotI; S, SpeI; St, StuI; X, XhoI.

b, DNA isolated from ES cell clones was digested with SpeI and analyzedby Southern blot hybridization with both, a 0.7 kb external genomicfragment (probe 1, left), and a 0.5 kb cDNA fragment (probe 2, right).Sizes are in kilobases. In both, the 26 kb fragment is indicative of thewild-type allele, while the 20 kb and the 7.8 kb fragments,respectively, originate from the targeted allele. Asterisks indicate twoclones positive for homologous recombination.

c, A pair of 2 days-old littermates, wild-type (upper) andPax4-deficient (lower), showing the reduced size of the null-mutantmouse.

d, Embryos were genotyped by PCR analysis using genomic DNA from yolksacs or tails, and two sets of primers (located as shown by arrows ina), in order to amplify wildtype-paired domain and/or, the neo gene.

FIG. 2 (FIGS. 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h). Analysis of LacZactivity, insulin and glucagon expression, in the pancreas of Pax4 (+/-)and Pax4 (-/-) E16.5 embryos and newborn mice

In E16.5 (+/-) pancreas, insulin (arrow in a) and glucagon (arrow in b)cells are associated with regions where LacZ activity (arrowheads in aand b) is detected. In contrast, in the pancreas of E16.5 (-/-) embryos,expression of LacZ (arrowheads in e) is diminished, whereas insulin isabsent (compare a and e). In E16.5 (-/-) pancreas a larger number ofglucagon-cells is found (arrow in f), associated with regions of LacZactivity (arrowhead in f). In (+/-) newborn pancreas, LacZ expression isrestricted to insulin cells (arrow in c), and appears surrounded by theglucagon-cells (arrow in d). In (-/-) newborn pancreas, no expression ofLacZ activity or insulin is detected (compare c, d and g). Meanwhile, in(-/-) newborn pancreas, numerous glucagon-cells are present (arrow inh), also abnormally distributed in clusters (compare d and h).Magnification is 400×.

FIG. 3 (FIGS. 3a, 3b, 3c, 3d, 3e, 3f). Expression of Pdx1 andsomatostatin in Pax4 (+/+) and (-/-) newborn pancreas.

In the dorsal pancreas of both, (+/+) and (-/-) E10.5 embryos, the earlyexpression of Pdx1 looks similar (compare a and b).

In newborn (+/+) pancreas, Pdx1 is restricted to matureinsulin-producing β-cells (arrows in c). In the pancreas of Pax4 (-/-)newborn mice, no expression of Pdx1 is detected (compare c and d). In(+/+) newborn pancreas, somatostatin producing δ-cells are mainlydistributed within the cells that surround the Islets of Langerhans(arrows in e). In (-/-) newborn pancreas, however, somatostatin is notdetected (compare e and f). a and b are vibratome sections. c and d arecryostat sections. e and f are paraffin sections. Magnification in (c-f)is (200×), and in a and b is (400×).

FIG. 4 (FIGS. 4a, 4b, 4c, 4d). Histological analysis of Pax4 (+/+) and(-/-) pancreas from newborn mice, and expression of α-amylase

Exocrine acini are shown in (a), containing groups of pyramidally-shapedcells (arrowheads), with their nuclei at the base. Exocrine cells arealso present in newborn (-/-) pancreas (arrowheads). However, extensiveareas of cytoplasm are observed, as well as a disorganized distributionof their nuclei (compare a and b). In the (+/+) newborn pancreas,exocrine cells contain different amounts of α-amylase (arrows andarrowhead in ac), reflecting the depletion of digestive enzymes thatnormally follows suckling. In almost all the exocrine tissue of newborn(-/-) pancreas, however, a large amount of α-amylase is present (arrowsin e), indicating that its secretion might be affected. Magnification ina and b is (400×), and in c and d is (200×).

FIG. 5. Model for the role of Pax4 in endocrine differentiation, in themouse pancreas.

Earliest endocrine progenitor cells are characterized by theirexpression of Pdx1, at around E8.5 of mouse development (Guz et al.,Development 121 (1995), 11-18). Differentiation of α-cells occurs veryearly in development, in precursors in which presumably Pdx1 is nolonger present. In the earliest β-cell progenitors, genes like Pdx1 andinsulin are selectively expressed, and maintenance of their expressionseems important for their progression into differentiation. In matureendocrine cells: Pdx1, insulin and Pax4 have their expression restrictedto the β-lineage. Pax4 may promote and/or maintain the expression ofgenes specific for the insulin-producing β-cells.

FIGS. 6 to 21. Sequences 1 to 15 (identified below by SEQ ID No.).

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES

Antibodies used against insulin, glucagon and amylase are commerciallyavailable from several companies. The antibodies used in Applicants'experiments were purchased from Boeringer Mannheim. The Pdx1 antibodywas a generous gift from Prof. Thomas Edlund (Department ofMicrobiology, University of Umea, S-901 87 Umea, Sweden). Antibodieswith the same specificity i.e. which specifically recognize the Pdx1protein can be prepared using the Pdx1 protein as an antigen accordingto conventional procedures.

EXAMPLE 1 Preparation of Construct for Homologous Recombination andGeneration of Pax4-Deficient Mice

Targeted deletion of almost all the entire paired box domain (darkboxes; exons 2, 3 and 4) of Pax4 was produced, by fusing in frame aβ-galactosidase-neomycin resistance cassette. A 5.1 kb Xba-Xhol fragmentcontaining LacZpA-pGKNeopA sequences was blunt ended and ligated intothe Nhel-digested and blunt ended Pax4 construct. The LacZpA-pGKNeopAconstruct was made by combining the pGKneo plasmid (Soriano et al.,1991, Cell 64:693-702) and the β-galactosidase gene which is availablefrom several commercial sources (e.g. pCH110 vector from Pharmacia). The5' Nhel site was retained and a new Spel site was generated at the 3'end. A F9 polyoma early promoter derived HSV TK gene was added upstreamto the 5' homology, for negative selection. R1 ES-cells wereelectroporated and selected following standard procedures. Positiveclones were used to generate chimeras by morula aggregation. Tail andyolk sac DNA-isolation and PCR amplification of genomic DNA wereperformed using standard procedures. Analysis of the phenotype was donein NMR1 and C57BL/6 mice. No differences were observed between bothgenetic backgrounds.

EXAMPLE 2 Studies on the Expression of Pax4 During Development

A genomic screen has previously identified Pax4 as a member of themurine family of Pax genes (Walther et al., Genomics 11 (1991),424-434). By RT-PCR Applicants isolated the corresponding cDNA sequence.

PCR amplification of a fragment (615 nt) encoding the paired andhomeobox domains of Pax4 cDNA was done with 1 mg of total RNA isolatedfrom E13.5 mouse embryos. Reverse transcription was performed with a kitfrom Pharmacia, and random primers. Primers used were obtained aftersequencing the corresponding genomic regions encoding the paired (5',sense primer: 5' AGC AAT AAG AGG GAT GCG ACC 3' (SEQ ID No. 5)) andhomeobox (3', antisense: 5' AGC TGT GCT TCC CAT TTC AGC 3' (SEQ ID No.6)) domains, respectively. In all the PCR reactions that were performed(including 5' and 3' RACE), Taq polymerase was added after the firstdenaturation step (95° C., 3 min), before cycling proceeded. 2 units ofTaq polymerase together with 1 unit of "Perfect Match" (from Stratagene)were added. 35 cycles were performed: 95° C., 11/2 min; 60° C. 11/2 min;72° C. 21/2 min. A final step with: 60° C., 11/2 min and 72° C. for 10min was performed. A faint band with the expected size was seen aftergel electrophoresis in 2% low-melting agarose. This was excised andreamplified (5 ml of molten agarose) with the same program. DNA waselectroeluted from a 2% preparative gel, filled-in and blunt-end clonedinto Bluescript digested with SmaI, following standard procedures.Sequencing of the isolated clones was done with a sequencing kit fromPharmacia. 5'-RACE amplification was performed using a kit fromGIBCO-BRL, following manufacturer's instructions. Briefly, 5 mg of totalRNA isolated from the posterior region of E10.5 mouse embryos (posteriorto the hindlimb bud: "tail-RNA") was used as template for first-strandcDNA synthesis. Two sets of nested primers were used in combination withthe AP (primer BS41: 5' GCG AAT TCC CTG AAG TGC CCG AAG TAC TCG ATT 3'(SEQ ID No. 7)) and UAP (primer BS57: 5' GGC TCC GTG AAA TGT CAC AG 3'(SEQ ID No. 8)) primers, respectively. In the first PCR reaction, oneprimer specific for Pax4 located close to the 5' region of its knownsequence was used in conjunction with the "AP" primer 5° CGT AGT ACT GTCGAC TAG CAG GGI IGG GII GGG IIG 3' (SEQ ID No. 9) from the commerciallyavailable kit (5' Race system from GIBCO-BRL). The "AP" primer containsan adaptor linked to a dG-tail which anneals to the dC-tailed cDNA. Forthe second reaction, a "nested" Pax4 primer ("nested meaning locatedmore 5' or `upstream` to the Pax4 primer used in the first reaction) wasused together with the commercial "UAP" primer (primer that containsonly the adaptor sequence of the AP primer 5° CGT AGT ACT GTC GAC TAG CA3' (SEQ ID No. 10). First PCR (AP/BS41 primers) was performed with 5 mlof dC-tailed cDNA (35 cycles of: 94° C., 11/2 min; 55° C., 11/2 min; 72°C., 3 min). 2 ml of this resulting PCR reaction (diluted 20 times) werereamplified with primers UAP/BS57, and 40 cycles of: 94° C., 1 min; 60°C., 1 min; 72° C., 21/2 min. A single band of approximately 500 nt wasseen after gel electrophoresis. This was electroeluted and cloned intothe TA-pGEM vector (PROMEGA). 180 nt of 5' new sequence information wasobtained with this approach. 3' RACE amplification was done using 2.5 mgof "tail" E10.5 RNA as template. First-strand cDNA synthesis wasperformed with the NotI-oligo dT primer provided by the First-strandcDNA synthesis kit (Pharmacia). First PCR was performed with the NotIprimer (5' AAC TGG AAG AAT TCG CGG CCG CAG GAA 3' (SEQ ID No. 11)) andBS36 (5° CAG GAA GAC CAG AGC TTG CAC TGG 3' (SEQ ID No. 12)) primers. 35cycles (94° C., 11/2 min; 55° C., 11/2 min; 72° C., 3 min) wereperformed. 2 ml of this PCR reaction (diluted 20 times) were reamplifiedwith a nested primer (BS42: 5' GCG GAT CCC ACA GGA ATC GGG CTA TCT TC 3'(SEQ ID No. 13)) and NotI primer. After gel electrophoresis, a prominentband of approximately 550 nt was excised from a 2% low-melting agarosegel. 2 ml of molten agarose were reamplified with another nested primer(BS59: 5' GCG CAG GCA AGA GAA GCT GA 3' (SEQ ID No. 14)) and NotIprimer. The last two PCRs were: 40 cycles of: 60° C., 11/2 min; 72° C.,3 min; 94° C., 11/2 min. A 420 nt prominent band was electroeluted andsubcloned into TA-pGEM vector. The fidelity of the amplified 5' and3'-PCR fragments was assessed by two procedures: first, a 1.1 kbfragment was amplified from "tail" E10.5 RNA, using primers (senseprimer: 5' CTT CCA GAA GGA GCT CTC 3' (SEQ ID No. 15) and its antisenseprimer: 5' TGG GAT GAT GGC ACT TGT C 3' (SEQ ID No. 16)) designed fromthe most 5' and 3' newly sequences. Its size and sequence were asexpected. Also, the sequence of the three PCR-isolated fragments (5'-,615 nt and 3'-) was compared with the equivalent coding regions in agenomic clone.

Analysis of Pax4 expression revealed that its transcripts are restrictedto a few cells in the ventral spinal cord and the early developingpancreas. In order to investigate the function of this gene duringdevelopment, Applicants generated Pax4-deficient mice; see Example 1.Inactivation of Pax4 after homologous recombination in ES-cells wasachieved, after deleting almost the entire paired box domain, and fusingthe β-galactosidase gene in frame with the amino-terminus of Pax4 (FIG.1a). This approach also allowed Applicants to analyze in more detail theexpression of Pax4 throughout development, by detection of LacZactivity.

Heterozygous mice do not exhibit any obvious abnormalities and they areviable and fertile. Staining of (+/-) embryos at day 10.5 of development(E10.5), revealed LacZ activity in cells within the dorsal pancreas. Fordetection of LacZ activity, X-gal staining of mouse embryos and isolatednewborn pancreas was performed, following standard procedures. Apost-fixation was performed in 4% paraformaldehyde, at 4° C., overnight.For immunohistochemistry, X-gal stained embryos and tissues wereembedded in paraffin, and sectioned (10 μM) with a microtome. Expressionof LacZ in the pancreas of Pax4 (+/-) embryos proceeded until birth. Inheterozygous newborn pancreas, LacZ activity was found restricted todiscrete areas corresponding to the β-cells in the Islets of Langerhans,as judged by co-expression of LacZ and insulin (FIGS. 1c and d).

Pax4-deficient offspring were born with the expected Mendeliandistribution, indicating that the absence of Pax4 is not lethal inutero. At birth, Pax4 (-/-) mice appear normal and wereindistinguishable from their littermates. However, 48 hours later theyexhibited growth retardation and dehydration (FIG. 1c). AllPax4-deficient mice died within the first three days after birth,demonstrating the complete penetrance of the mutant phenotype. Pancreasof newborn Pax4 (-/-) mice showed a normal macroscopical appearance. Theexpression of LacZ in the pancreas of Pax4 (-/-) mutant mice was alsoinvestigated. In E10.5 (-/-) embryos LacZ activity was detected in cellsof the dorsal pancreas, in a similar manner to heterozygous embryos. Nodifferences in the expression of LacZ activity were observed in thedeveloping pancreas of Pax4 (+/-) and (-/-) embryos, until approximatelyE16.5. After this, however, in the pancreas of Pax4-deficient mice LacZexpression began to diminish (compare FIGS. 2a, b and 2e, f), and itbecame undetectable after birth (compare FIGS. 2d and 2g).

EXAMPLE 3 Expression of Insulin and Glucagon in Pax4 Targeted Mice

In the pancreas, all endocrine cells arise from common multipotentprecursors (Alpert, Cell 53 (1988), 295-308). The first precursor cellscontaining insulin and glucagon, appear around E9.5 (Gittes et al.,Proc. Natl. Acad. Sci. USA 89 (1992), 1128-1132; Teitelman et al.,Development 118 (1993), 1031-1039). In the mouse pancreas,differentiation of exocrine and most endocrine cells starts around E16.5of development (Githens, The Pancreas: biology, pathobiology, anddisease. Second Edition (ed. Vay Liang W. Go, et al.) Raven, N.Y.,(1993), 21-73). At birth, insulin production is mostly restricted tofully differentiated β-cells, located in the center of the islets ofLangerhans. These are surrounded by the glucagon-producing α-cells.β-cells comprise the majority of the endocrine population, whereas theα-cells represent only a small fraction (Slack, Development 121 (1995),1569-1580). Applicants have tested for the expression of insulin andglucagon in the pancreas of Pax4 heterozygous and Pax4 null-mutant mice,by immunochemistry on paraffin sections performed as previouslydescribed (Oliver et al., EMBO J. 7 (1988), 3199-3209).

At E10.5, insulin-producing cells were detected in the pancreas of Pax4(+/-) and (-/-) embryos. In E16.5 heterozygous embryos, all insulincells were found included within the area of LacZ activity (FIG. 2a). Aspreviously mentioned, in heterozygous newborn mice LacZ and insulin weredetected in the same cells (FIG. 2c), indicating that later indevelopment Pax4 expression is restricted to the insulin-producingβ-cells. In contrast, in the pancreas of Pax4 (-/-) E16.5 embryos andnewborn mice, few if any insulin-producing cells were detected (FIG. 2e,g). This indicates that in the absence of Pax4, the maturation of thepancreatic β-cells was affected. In the pancreas of E10.5 (+/-) and(-/-) embryos, cells containing glucagon are present. In heterozygousE16.5 embryos, glucagon was detected in cells, most of which werecontained within the area of LacZ activity (FIG. 2b). In newbornheterozygous pancreas, glucagon was present in cells surrounding thearea where the LacZ was expressed, but were not included within it (FIG.2d). In the pancreas of Pax4 (-/-) E16.5 embryos and newborn mice, aconsiderably larger number of glucagon-producing cells was found. Thesecells also showed an aberrantly clustered distribution (FIGS. 2f and h).

EXAMPLE 4 Expression of Pdx1 in Pax4 Targeted Mice

In order to further confirm that in the pancreas of Pax4-deficientnewborn mice β-cells are missing, the expression of a specific β-cellmarker, Pdx1, was tested. This gene appears very early in pancreasdevelopment (most likely in the earliest pancreatic progenitors), butlater becomes restricted to the fully differentiated β-cells (Guz etal., Development 121, 11-18 (1995); Miller et al., EMBO J. 13 (1994),1145-1156) (FIG. 3c). Its early expression was assayed by whole-mountimmunostaining of E10.5 mouse embryos, using an established protocol(Ohlsson et al., EMBO J. 13 (1994), 1145-1158), except that bleachingwas for 24 hours. After staining, E10.5 embryos were postfixed, embeddedin a gelatin-BSA matrix and sectioned with a vibratome. Expression ofPdx1 in newborn pancreas by immunostaining on cryostat sections wasperformed as previously described, with the following modifications:newborn pancreas was fixed 3 hours with 4% paraformaldehyde, and thencryoprotected with 30% sucrose in PBS, overnight. After washing-off thesecondary antibody, secretions were incubated with 0.6% H₂ O₂, 20 min.At E10.5, Pdx1 was similarly expressed in the pancreas of both, Pax4(+/+) and (-/-) embryos (FIGS. 3a,b). However, in the pancreas ofPax4-deficient newborn mice, Pdx1 expression was not detected. Thisconfirms the initial conclusion that, in the pancreas of Pax4null-mutant mice, the mature β-cells are absent. It also suggests that,although Pax4 may not be required for generation of the earliestendocrine precursors, it is crucial for β-cell differentiation.

EXAMPLE 5 Expression of Somatostatin in Pax4 Targeted Mice

During mouse embryogenesis, somatostatin-producing cells start todifferentiate later than α- and β-cells (Teitelman et al., Development118 (1993), 1031-1039). In the pancreas of Pax4 (+/+) newborn nice,somatostatin-producing δ-cells were mainly distributed in the peripheryof islets, intermingled with the α-cells (FIG. 3e), suggesting thatinactivation of Pax4 also affected the maturation of the δ-lineage.Expression of somatostatin in the gut, however, appeared unaffected.

EXAMPLE 6 Histological Analysis of Newborn Pancreas From Pax4 TargetedMice

Exocrine pancreas is a lobulated, branched, acinar gland, withpyramidal-shaped secretory cells and basal nuclei (Pictet, Devl. Biol.29 (1972), 436-436) (FIG. 4a). Histological analysis of newborn Pax4(-/-) pancreas showed that in this gland, exocrine tissue was present.However, the cytoplasm of exocrine cells seemed expanded, and theirnuclei did not show a homogenously basal distribution (FIG. 4b). Whenpancreas of 3-days old wildtype mice was analyzed for the presence of aspecific exocrine enzyme (α-amylase), large portions of exocrine tissueshowed little or no expression of such marker (FIG. 4c). This mostlikely reflects the depletion of exocrine digestive enzymes thatnormally occurs after suckling (Githens, The Pancreas: biology,pathobiology, and disease. Second Edition (ed. Vay Liang W. Go, et al.)Raven, N.Y., (1993), 21-73). At birth, Pax4-deficient mice are able tosuckle, since their stomachs are full with milk (FIG. 1c). However,3-days old Pax4 (-/-) pancreas showed a strong expression of α-amylasein all exocrine cells, indicating that they might not be able to secretetheir enzymes into the digestive tract (FIG. 4d).

The conclusions to be drawn from the results of the above examples withrespect to the influence of the Pax4 and Pdx1 genes on the developmentof pancreatic cells is shown in FIG. 5.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope thereof.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 16                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1275 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION:166..1161                                              #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ACCAGCAACC CTGGAGCCTG CACAGACCCT GAGACCTCTT CCTGAATTCC CA - #CCTTTTTT         60                                                                          - CCTCCATCCA GTACCAGTCC CAAAGAGAAA CTTCCAGAAG GAGCTCTCCG TT - #TTCAGTTT        120                                                                          #CAG CAG        174TCCT TCTGCGAGGA GTACCAGTGT GAAGC ATG                       #              Met Gln G - #ln                                                #                1                                                            - GAC GGA CTC AGC AGT GTG AAT CAG CTA GGG GG - #A CTC TTT GTG AAT GGC          222                                                                          Asp Gly Leu Ser Ser Val Asn Gln Leu Gly Gl - #y Leu Phe Val Asn Gly           #      15                                                                     - CGG CCC CTT CCT CTG GAC ACC AGG CAG CAG AT - #T GTG CAG CTA GCA ATA          270                                                                          Arg Pro Leu Pro Leu Asp Thr Arg Gln Gln Il - #e Val Gln Leu Ala Ile           # 35                                                                          - AGA GGG ATG CGA CCC TGT GAC ATT TCA CGG AG - #C CTT AAG GTA TCT AAT          318                                                                          Arg Gly Met Arg Pro Cys Asp Ile Ser Arg Se - #r Leu Lys Val Ser Asn           #                 50                                                          - GGC TGT GTG AGC AAG ATC CTA GGA CGC TAC TA - #C CGC ACA GGT GTC TTG          366                                                                          Gly Cys Val Ser Lys Ile Leu Gly Arg Tyr Ty - #r Arg Thr Gly Val Leu           #             65                                                              - GAA CCC AAG TGT ATT GGG GGA AGC AAA CCA CG - #T CTG GCC ACA CCT GCT          414                                                                          Glu Pro Lys Cys Ile Gly Gly Ser Lys Pro Ar - #g Leu Ala Thr Pro Ala           #         80                                                                  - GTG GTG GCT CGA ATT GCC CAG CTA AAG GAT GA - #G TAC CCT GCT CTT TTT          462                                                                          Val Val Ala Arg Ile Ala Gln Leu Lys Asp Gl - #u Tyr Pro Ala Leu Phe           #     95                                                                      - GCC TGG GAG ATC CAA CAC CAG CTT TGC ACT GA - #A GGG CTT TGT ACC CAG          510                                                                          Ala Trp Glu Ile Gln His Gln Leu Cys Thr Gl - #u Gly Leu Cys Thr Gln           100                 1 - #05                 1 - #10                 1 -       #15                                                                           - GAC AAG GCT CCC AGT GTG TCC TCT ATC AAT CG - #A GTA CTT CGG GCA CTT          558                                                                          Asp Lys Ala Pro Ser Val Ser Ser Ile Asn Ar - #g Val Leu Arg Ala Leu           #               130                                                           - CAG GAA GAC CAG AGC TTG CAC TGG ACT CAA CT - #C AGA TCA CCA GCT GTG          606                                                                          Gln Glu Asp Gln Ser Leu His Trp Thr Gln Le - #u Arg Ser Pro Ala Val           #           145                                                               - TTG GCT CCA GTT CTT CCC AGT CCC CAC AGT AA - #C TGT GGG GCT CCC CGA          654                                                                          Leu Ala Pro Val Leu Pro Ser Pro His Ser As - #n Cys Gly Ala Pro Arg           #       160                                                                   - GGC CCC CAC CCA GGA ACC AGC CAC AGG AAT CG - #G GCT ATC TTC TCC CCG          702                                                                          Gly Pro His Pro Gly Thr Ser His Arg Asn Ar - #g Ala Ile Phe Ser Pro           #   175                                                                       - GGA CAA GCC GAG GCA CTG GAG AAA GAG TTT CA - #G CGT GGG CAG TAT CCA          750                                                                          Gly Gln Ala Glu Ala Leu Glu Lys Glu Phe Gl - #n Arg Gly Gln Tyr Pro           180                 1 - #85                 1 - #90                 1 -       #95                                                                           - GAT TCA GTG GCC CGT GGG AAG CTG GCT GCT GC - #C ACC TCT CTG CCT GAA          798                                                                          Asp Ser Val Ala Arg Gly Lys Leu Ala Ala Al - #a Thr Ser Leu Pro Glu           #               210                                                           - GAC ACG GTG AGG GTT TGG TTT TCT AAC AGA AG - #A GCC AAA TGG CGC AGG          846                                                                          Asp Thr Val Arg Val Trp Phe Ser Asn Arg Ar - #g Ala Lys Trp Arg Arg           #           225                                                               - CAA GAG AAG CTG AAA TGG GAA GCA CAG CTG CC - #A GGT GCT TCC CAG GAC          894                                                                          Gln Glu Lys Leu Lys Trp Glu Ala Gln Leu Pr - #o Gly Ala Ser Gln Asp           #       240                                                                   - CTG ACG ATA CCA AAA AAT TCT CCA GGG ATC AT - #C TCT GCA CAG CAG TCC          942                                                                          Leu Thr Ile Pro Lys Asn Ser Pro Gly Ile Il - #e Ser Ala Gln Gln Ser           #   255                                                                       - CCC GGC AGT GTA CCC TCA GCT GCC TTG CCT GT - #G CTG GAA CCA TTG AGT          990                                                                          Pro Gly Ser Val Pro Ser Ala Ala Leu Pro Va - #l Leu Glu Pro Leu Ser           260                 2 - #65                 2 - #70                 2 -       #75                                                                           - CCT TCC TTC TGT CAG CTA TGC TGT GGG ACA GC - #A CCA GGC AGA TGT TCC         1038                                                                          Pro Ser Phe Cys Gln Leu Cys Cys Gly Thr Al - #a Pro Gly Arg Cys Ser           #               290                                                           - AGT GAC ACC TCA TCC CAG GCC TAT CTC CAA CC - #C TAC TGG GAC TGC CAA         1086                                                                          Ser Asp Thr Ser Ser Gln Ala Tyr Leu Gln Pr - #o Tyr Trp Asp Cys Gln           #           305                                                               - TCC CTC CTT CCT GTG GCT TCC TCC TCA TAT GT - #G GAA TTT GCC TGC CCT         1134                                                                          Ser Leu Leu Pro Val Ala Ser Ser Ser Tyr Va - #l Glu Phe Ala Cys Pro           #       320                                                                   - GCC TCA CCA CCC ATC CTG TGC ATC ATC TGATTGGAG - #G CCCAGGACAA               1181                                                                          Ala Ser Pro Pro Ile Leu Cys Ile Ile                                           #   330                                                                       - GTGCCATCAT CCCATTGCTC AAACTGGCCA TAAGACACCT CTATTTGACA GT - #AATAAAAA       1241                                                                          #      1275        AAAA AAAAAAAAGG GGGG                                       - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 332 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Gln Gln Asp Gly Leu Ser Ser Val Asn Gl - #n Leu Gly Gly Leu Phe         #                 15                                                          - Val Asn Gly Arg Pro Leu Pro Leu Asp Thr Ar - #g Gln Gln Ile Val Gln         #             30                                                              - Leu Ala Ile Arg Gly Met Arg Pro Cys Asp Il - #e Ser Arg Ser Leu Lys         #         45                                                                  - Val Ser Asn Gly Cys Val Ser Lys Ile Leu Gl - #y Arg Tyr Tyr Arg Thr         #     60                                                                      - Gly Val Leu Glu Pro Lys Cys Ile Gly Gly Se - #r Lys Pro Arg Leu Ala         # 80                                                                          - Thr Pro Ala Val Val Ala Arg Ile Ala Gln Le - #u Lys Asp Glu Tyr Pro         #                 95                                                          - Ala Leu Phe Ala Trp Glu Ile Gln His Gln Le - #u Cys Thr Glu Gly Leu         #           110                                                               - Cys Thr Gln Asp Lys Ala Pro Ser Val Ser Se - #r Ile Asn Arg Val Leu         #       125                                                                   - Arg Ala Leu Gln Glu Asp Gln Ser Leu His Tr - #p Thr Gln Leu Arg Ser         #   140                                                                       - Pro Ala Val Leu Ala Pro Val Leu Pro Ser Pr - #o His Ser Asn Cys Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Pro Arg Gly Pro His Pro Gly Thr Ser Hi - #s Arg Asn Arg Ala Ile         #               175                                                           - Phe Ser Pro Gly Gln Ala Glu Ala Leu Glu Ly - #s Glu Phe Gln Arg Gly         #           190                                                               - Gln Tyr Pro Asp Ser Val Ala Arg Gly Lys Le - #u Ala Ala Ala Thr Ser         #       205                                                                   - Leu Pro Glu Asp Thr Val Arg Val Trp Phe Se - #r Asn Arg Arg Ala Lys         #   220                                                                       - Trp Arg Arg Gln Glu Lys Leu Lys Trp Glu Al - #a Gln Leu Pro Gly Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ser Gln Asp Leu Thr Ile Pro Lys Asn Ser Pr - #o Gly Ile Ile Ser Ala         #               255                                                           - Gln Gln Ser Pro Gly Ser Val Pro Ser Ala Al - #a Leu Pro Val Leu Glu         #           270                                                               - Pro Leu Ser Pro Ser Phe Cys Gln Leu Cys Cy - #s Gly Thr Ala Pro Gly         #       285                                                                   - Arg Cys Ser Ser Asp Thr Ser Ser Gln Ala Ty - #r Leu Gln Pro Tyr Trp         #   300                                                                       - Asp Cys Gln Ser Leu Leu Pro Val Ala Ser Se - #r Ser Tyr Val Glu Phe         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ala Cys Pro Ala Ser Pro Pro Ile Leu Cys Il - #e Ile                         #               330                                                           - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2481 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION:163..1470                                              #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ACAACGACGA AAGAGAGGAT GCCTCTTAAA GGCAGAAGAC TTTAACCAAG GG - #CGGTGAGC         60                                                                          - AGATGTGTGA GATCTTCTAT TCTAGAAGTG GACGTATATC CCAGTTCTCA GA - #GCCCCGTA        120                                                                          - TTCGAGCCCC GTGGGATCCG GAGGCTGCCA ACCAGCTCCA GC ATG CAG - # AAC AGT           174                                                                          #           Met Gln Asn Ser                                                   #             1                                                               - CAC AGC GGA GTG AAT CAG CTT GGT GGT GTC TT - #T GTC AAC GGG CGG CCA          222                                                                          His Ser Gly Val Asn Gln Leu Gly Gly Val Ph - #e Val Asn Gly Arg Pro           #  20                                                                         - CTG CCG GAC TCC ACC CGG CAG AAG ATC GTA GA - #G CTA GCT CAC AGC GGG          270                                                                          Leu Pro Asp Ser Thr Arg Gln Lys Ile Val Gl - #u Leu Ala His Ser Gly           #                 35                                                          - GCC CGG CCG TGC GAC ATT TCC CGA ATT CTG CA - #G ACC CAT GCA GAT GCA          318                                                                          Ala Arg Pro Cys Asp Ile Ser Arg Ile Leu Gl - #n Thr His Ala Asp Ala           #             50                                                              - AAA GTC CAG GTG CTG GAC AAT GAA AAC GTA TC - #C AAC GGT TGT GTG AGT          366                                                                          Lys Val Gln Val Leu Asp Asn Glu Asn Val Se - #r Asn Gly Cys Val Ser           #         65                                                                  - AAA ATT CTG GGC AGG TAT TAC GAG ACT GGC TC - #C ATC AGA CCC AGG GCA          414                                                                          Lys Ile Leu Gly Arg Tyr Tyr Glu Thr Gly Se - #r Ile Arg Pro Arg Ala           #     80                                                                      - ATC GGA GGG AGT AAG CCA AGA GTG GCG ACT CC - #A GAA GTT GTA AGC AAA          462                                                                          Ile Gly Gly Ser Lys Pro Arg Val Ala Thr Pr - #o Glu Val Val Ser Lys           #100                                                                          - ATA GCC CAG TAT AAA CGG GAG TGC CCT TCC AT - #C TTT GCT TGG GAA ATC          510                                                                          Ile Ala Gln Tyr Lys Arg Glu Cys Pro Ser Il - #e Phe Ala Trp Glu Ile           #               115                                                           - CGA GAC AGA TTA TTA TCC GAG GGG GTC TGT AC - #C AAC GAT AAC ATA CCC          558                                                                          Arg Asp Arg Leu Leu Ser Glu Gly Val Cys Th - #r Asn Asp Asn Ile Pro           #           130                                                               - AGT GTG TCA TCA ATA AAC AGA GTT CTT CGC AA - #C CTG GCT AGC GAA AAG          606                                                                          Ser Val Ser Ser Ile Asn Arg Val Leu Arg As - #n Leu Ala Ser Glu Lys           #       145                                                                   - CAA CAG ATG GGC GCA GAC GGC ATG TAT GAT AA - #A CTA AGG ATG TTG AAC          654                                                                          Gln Gln Met Gly Ala Asp Gly Met Tyr Asp Ly - #s Leu Arg Met Leu Asn           #   160                                                                       - GGG CAG ACC GGA AGC TGG GGC ACA CGC CCT GG - #T TGG TAT CCC GGG ACT          702                                                                          Gly Gln Thr Gly Ser Trp Gly Thr Arg Pro Gl - #y Trp Tyr Pro Gly Thr           165                 1 - #70                 1 - #75                 1 -       #80                                                                           - TCA GTA CCA GGG CAA CCC ACG CAA GAT GGC TG - #C CAG CAA CAG GAA GGA          750                                                                          Ser Val Pro Gly Gln Pro Thr Gln Asp Gly Cy - #s Gln Gln Gln Glu Gly           #               195                                                           - GGG GGA GAG AAC ACC AAC TCC ATC AGT TCT AA - #C GGA GAA GAC TCG GAT          798                                                                          Gly Gly Glu Asn Thr Asn Ser Ile Ser Ser As - #n Gly Glu Asp Ser Asp           #           210                                                               - GAA GCT CAG ATG CGA CTT CAG CTG AAG CGG AA - #G CTG CAA AGA AAT AGA          846                                                                          Glu Ala Gln Met Arg Leu Gln Leu Lys Arg Ly - #s Leu Gln Arg Asn Arg           #       225                                                                   - ACA TCT TTT ACC CAA GAG CAG ATT GAG GCT CT - #G GAG AAA GAG TTT GAG          894                                                                          Thr Ser Phe Thr Gln Glu Gln Ile Glu Ala Le - #u Glu Lys Glu Phe Glu           #   240                                                                       - AGG ACC CAT TAT CCA GAT GTG TTT GCC CGG GA - #A AGA CTA GCA GCC AAA          942                                                                          Arg Thr His Tyr Pro Asp Val Phe Ala Arg Gl - #u Arg Leu Ala Ala Lys           245                 2 - #50                 2 - #55                 2 -       #60                                                                           - ATA GAT CTA CCT GAA GCA AGA ATA CAG GTA TG - #G TTT TCT AAT CGA AGG          990                                                                          Ile Asp Leu Pro Glu Ala Arg Ile Gln Val Tr - #p Phe Ser Asn Arg Arg           #               275                                                           - GCC AAA TGG AGA AGA GAA GAG AAA CTG AGG AA - #C CAG AGA AGA CAG GCC         1038                                                                          Ala Lys Trp Arg Arg Glu Glu Lys Leu Arg As - #n Gln Arg Arg Gln Ala           #           290                                                               - AGC AAC ACT CCT AGT CAC ATT CCT ATC AGC AG - #C AGC TTC AGT ACC AGT         1086                                                                          Ser Asn Thr Pro Ser His Ile Pro Ile Ser Se - #r Ser Phe Ser Thr Ser           #       305                                                                   - GTC TAC CAG CCA ATC CCA CAG CCC ACC ACA CC - #T GTC TCC TCC TTC ACA         1134                                                                          Val Tyr Gln Pro Ile Pro Gln Pro Thr Thr Pr - #o Val Ser Ser Phe Thr           #   320                                                                       - TCA GGT TCC ATG TTG GGC CGA ACA GAC ACC GC - #C CTC ACC AAC ACG TAC         1182                                                                          Ser Gly Ser Met Leu Gly Arg Thr Asp Thr Al - #a Leu Thr Asn Thr Tyr           325                 3 - #30                 3 - #35                 3 -       #40                                                                           - AGT GCT TTG CCA CCC ATG CCC AGC TTC ACC AT - #G GCA AAC AAC CTG CCT         1230                                                                          Ser Ala Leu Pro Pro Met Pro Ser Phe Thr Me - #t Ala Asn Asn Leu Pro           #               355                                                           - ATG CAA CCC CCA GTC CCC AGT CAG ACC TCC TC - #A TAC TCG TGC ATG CTG         1278                                                                          Met Gln Pro Pro Val Pro Ser Gln Thr Ser Se - #r Tyr Ser Cys Met Leu           #           370                                                               - CCC ACC AGC CCG TCA GTG AAT GGG CGG AGT TA - #T GAT ACC TAC ACC CCT         1326                                                                          Pro Thr Ser Pro Ser Val Asn Gly Arg Ser Ty - #r Asp Thr Tyr Thr Pro           #       385                                                                   - CCG CAC ATG CAA ACA CAC ATG AAC AGT CAG CC - #C ATG GGC ACC TCG GGG         1374                                                                          Pro His Met Gln Thr His Met Asn Ser Gln Pr - #o Met Gly Thr Ser Gly           #   400                                                                       - ACC ACT TCA ACA GGA CTC ATT TCA CCT GGA GT - #G TCA GTT CCC GTC CAA         1422                                                                          Thr Thr Ser Thr Gly Leu Ile Ser Pro Gly Va - #l Ser Val Pro Val Gln           405                 4 - #10                 4 - #15                 4 -       #20                                                                           - GTT CCC GGG AGT GAA CCT GAC ATG TCT CAG TA - #C TGG CCT CGA TTA CAG         1470                                                                          Val Pro Gly Ser Glu Pro Asp Met Ser Gln Ty - #r Trp Pro Arg Leu Gln           #               435                                                           - TAAAGAGAGA AGGAGAGAGC ATGTGATCGA GAGAGGAAAT TGTGTTCACT CT - #GCCAATGA       1530                                                                          - CTATGTGGAC ACAGCAGTTG GGTATTCAGG AAAGAAAGAG AAATGGCGGT TA - #GAAGCACT       1590                                                                          - TCACTTTGTA ACTGTCCTGA ACTGGAGCCC GGGAATGGAC TAGAACCAAG GA - #CCTTGCGT       1650                                                                          - ACAGAAGGCA CGGTATCAGT TGGAACAAAT CTTCATTTTG GTATCCAAAC TT - #TTATTCAT       1710                                                                          - TTTGGTGTAT TATTTGTAAA TGGGCATTGG TATGTTATAA TGAAGAAAAG AA - #CAACACAG       1770                                                                          - GCTGTTGGAT CGCGGATCTG TGTTGCTCAT GTGGTTGTTT AAAGGAAACC AT - #GATCGACA       1830                                                                          - AGATTTGCCA TGGATTTAAG AGTTTTATCA AGATATATCA AATACTTCTC CC - #CATCTGTT       1890                                                                          - CATAGTTTAT GGACTGATGT TCCAAGTTTG TATCATTCCT TTGCATATAA TT - #GAACCTGG       1950                                                                          - GACAACACAC ACTAGATATA TGTAAAAACT ATCTGTTGGT TTTCCAAAGG TT - #GTTAACAG       2010                                                                          - ATGAAGTTTA TGTGCAAAAA AGGGTAAGAT ATGAATTCAA GGAGAAGTTG AT - #AGCTAAAA       2070                                                                          - GGTAGAGTGT GTCTTCGATA TAATACAATT TGTTTTATGT CAAAATGTAA GT - #ATTTGTCT       2130                                                                          - TCCCTAGAAA TCCTCAGAAT GATTTCTATA ATAAAGTTAA TTTCATTTAT AT - #TTGACAAG       2190                                                                          - AATACTCTAT AGATGTTTTA TACACATTTT CATGCAATCA TTTGTTTCTT TC - #TTGGCCAG       2250                                                                          - CAAAAGTTAA TTGTTCTTAG ATATAGCTGT ATTACTGTTC ACAGTCCAAT CA - #TTTTGTGC       2310                                                                          - ATCTAGAATT CATTCCTAAT CAATTAAAAG TGCTTGCAAG AGTTTTAAAC CT - #AAGTGTTT       2370                                                                          - TGCAGTTGTT CACAAATACA TATCAAAATT AACCATTGTT GATTGTAAAA AA - #AAAACCAT       2430                                                                          #           2481TATTTTC TTTATTACCC TTGACCGTAA GACATGAATT C                    - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 436 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Gln Asn Ser His Ser Gly Val Asn Gln Le - #u Gly Gly Val Phe Val         #                 15                                                          - Asn Gly Arg Pro Leu Pro Asp Ser Thr Arg Gl - #n Lys Ile Val Glu Leu         #             30                                                              - Ala His Ser Gly Ala Arg Pro Cys Asp Ile Se - #r Arg Ile Leu Gln Thr         #         45                                                                  - His Ala Asp Ala Lys Val Gln Val Leu Asp As - #n Glu Asn Val Ser Asn         #     60                                                                      - Gly Cys Val Ser Lys Ile Leu Gly Arg Tyr Ty - #r Glu Thr Gly Ser Ile         # 80                                                                          - Arg Pro Arg Ala Ile Gly Gly Ser Lys Pro Ar - #g Val Ala Thr Pro Glu         #                 95                                                          - Val Val Ser Lys Ile Ala Gln Tyr Lys Arg Gl - #u Cys Pro Ser Ile Phe         #           110                                                               - Ala Trp Glu Ile Arg Asp Arg Leu Leu Ser Gl - #u Gly Val Cys Thr Asn         #       125                                                                   - Asp Asn Ile Pro Ser Val Ser Ser Ile Asn Ar - #g Val Leu Arg Asn Leu         #   140                                                                       - Ala Ser Glu Lys Gln Gln Met Gly Ala Asp Gl - #y Met Tyr Asp Lys Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Arg Met Leu Asn Gly Gln Thr Gly Ser Trp Gl - #y Thr Arg Pro Gly Trp         #               175                                                           - Tyr Pro Gly Thr Ser Val Pro Gly Gln Pro Th - #r Gln Asp Gly Cys Gln         #           190                                                               - Gln Gln Glu Gly Gly Gly Glu Asn Thr Asn Se - #r Ile Ser Ser Asn Gly         #       205                                                                   - Glu Asp Ser Asp Glu Ala Gln Met Arg Leu Gl - #n Leu Lys Arg Lys Leu         #   220                                                                       - Gln Arg Asn Arg Thr Ser Phe Thr Gln Glu Gl - #n Ile Glu Ala Leu Glu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Lys Glu Phe Glu Arg Thr His Tyr Pro Asp Va - #l Phe Ala Arg Glu Arg         #               255                                                           - Leu Ala Ala Lys Ile Asp Leu Pro Glu Ala Ar - #g Ile Gln Val Trp Phe         #           270                                                               - Ser Asn Arg Arg Ala Lys Trp Arg Arg Glu Gl - #u Lys Leu Arg Asn Gln         #       285                                                                   - Arg Arg Gln Ala Ser Asn Thr Pro Ser His Il - #e Pro Ile Ser Ser Ser         #   300                                                                       - Phe Ser Thr Ser Val Tyr Gln Pro Ile Pro Gl - #n Pro Thr Thr Pro Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Ser Phe Thr Ser Gly Ser Met Leu Gly Ar - #g Thr Asp Thr Ala Leu         #               335                                                           - Thr Asn Thr Tyr Ser Ala Leu Pro Pro Met Pr - #o Ser Phe Thr Met Ala         #           350                                                               - Asn Asn Leu Pro Met Gln Pro Pro Val Pro Se - #r Gln Thr Ser Ser Tyr         #       365                                                                   - Ser Cys Met Leu Pro Thr Ser Pro Ser Val As - #n Gly Arg Ser Tyr Asp         #   380                                                                       - Thr Tyr Thr Pro Pro His Met Gln Thr His Me - #t Asn Ser Gln Pro Met         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Thr Ser Gly Thr Thr Ser Thr Gly Leu Il - #e Ser Pro Gly Val Ser         #               415                                                           - Val Pro Val Gln Val Pro Gly Ser Glu Pro As - #p Met Ser Gln Tyr Trp         #           430                                                               - Pro Arg Leu Gln                                                                     435                                                                   - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                CGAC C                                                     - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                TCAG C                                                     - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #         33       GCCC GAAGTACTCG ATT                                        - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               ACAG                                                       - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #       36         AGCA GGGNNGGGNN GGGNNG                                     - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               AGCA                                                       - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             27   GGCC GCAGGAA                                               - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24TGCA CTGG                                                  - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #            29    TCGG GCTATCTTC                                             - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               CTGA                                                       - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #  18              TC                                                         - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "oligonucleotide"                    #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 19               GTC                                                        __________________________________________________________________________

What is claimed is:
 1. A method for determining risk of developingjuvenile diabetes based upon absence of Pax4 mRNA or protein in a mammalcomprising:(a) determining the level or status of Pax4 mRNA in β-cellsof said mammal; and/or (b) determining the level or status of Pax4protein in β-cells of said mammal; and (c) comparing said level orstatus or Pax4 mRNA and/or Pax4 protein with the corresponding leveI innormal β-cells;wherein the term "level" denotes the amount of mRNA orprotein produced; and, the term "status" includes that the Pax4 gene,mRNA, protein or a transcription control element, including apromoter/enhancer sequence, may bear a mutation, deletion or any othermodifications which would affect the overall activity of the gene whencompared to the wild-type normal gene product, includingpost-translational modifications of the protein, and from the comparingdetermining an absence of Pax4 mRNA or protein and thus risk ofdeveloping juvenile diabetes.
 2. In a method for treating juvenilediabetes in a mammal comprising administering a medicament or treatmentfor a deficiency or failure of insulin production wherein theimprovement comprises:(I) determining risk of developing juvenilediabetes based upon absence of Pax4 mRNA or protein in a mammal by amethod comprising:(a) determining the level or status of Pax4 mRNA inβ-cells of said mammal; and/or (b) determining the level or status ofPax4 protein in β-cells of said mammal; and (c) comparing said level orstatus of Pax4 mRNA and/or Pax4 protein with the corresponding level innormal β-cells;wherein the term "level" denotes the amount of mRNA orprotein produced; and, the term "status" includes that the Pax4 gene,mRNA, protein or a transcription control element, including apromoter/enhancer sequence, may bear a mutation, deletion or any othermodifications which would affect the overall activity of the gene whencompared to the wild-type normal gene product, includingpost-translational modifications of the protein, and from the comparingdetermining an absence of Pax4 mRNA or protein and thus risk ofdeveloping juvenile diabetes prior to administering the medicament ortreatment if a risk of developing juvenile diabetes is indicated as aresult of step (I), to thereby treat juvenile diabetes.
 3. A method fortesting a medicament for or a gene therapy approach to juvenile diabetesbased on absence of Pax4 mRNA or protein comprising:(a') administeringthe medicament or the gene therapy; (a) determining the level or statusof Pax4 mRNA in pancreatic cells of said mammal; and/or (b) determiningthe level or status of Pax4 protein in pancreatic cells of said mammal;and (c) comparing said level or status of Pax4 mRNA and/or Pax4 proteinwith the corresponding level in normal pancreatic cells;wherein the term"level" denotes the amount of mRNA or protein produced; and, the term"status" includes that the Pax4 gene, mRNA, protein or a transcriptioncontrol element, including a promoter/enhancer sequence, may bear amutation, deletion or any other modifications which would affect theoverall activity of the gene when compared to the wild-type normal geneproduct, including post-translational modifications of the protein, andfrom the comparing determining an absence of Pax4 mRNA or protein andthus efficacy of the medicament or the gene therapy.
 4. The methodaccording to claim 1 further comprising(d) determining the level orstatus of Pax6 mRNA in pancreatic cells of said mammal; and/or (e)determining the level or status of Pax6 protein in pancreatic cells ofsaid mammal; and (f) comparing said level or status of Pax6 mRNA and/orPax6 protein with the corresponding level in normal pancreaticcells;wherein the term "level" denotes the amount of mRNA or proteinproduced; and, the term "status" includes that the Pax6 gene, mRNA,protein or a transcription control element, including apromoter/enhancer sequence, may bear a mutation, deletion or any othermodifications which would affect the overall activity of the gene whencompared to the wild-type normal gene product, includingpost-translational modifications of the protein, and from the comparingdetermining an absence of Pax6 mRNA or protein and thus risk ofdeveloping juvenile diabetes.
 5. The method of claim 1 wherein saidmammal is(i) an embryo; (ii) a newborn; or (iii) an adult.
 6. The methodaccording to claim 1 wherein said mammal is a mouse.
 7. The methodaccording to claim 1 wherein said mammal is a human.
 8. The methodaccording to claim 4, wherein steps (a) and/or (b) and optionally (d)and/or (e) are carried out in vivo.
 9. The method according to claims 4,wherein steps (a) and/or (b) and optionally (d) and/or (e) are carriedout in vitro.
 10. The method according to claim 4 wherein saiddetermination in step (a) and optionally in step (d) is effected byemploying(i) a nucleic acid sequence corresponding to at least a part ofthe Pax4 gene encoding at least part of the Pax4 protein and optionallya second nucleic acid sequence corresponding to at least a part of thePax6 gene encoding at least part of the Pax6 protein; (ii) a nucleicacid sequence complementary to the nucleic acid sequence(s) of (i); or(iii) a primer or a primer pair hybridizing to the nucleic acidsequence(s) of (i) or (ii).
 11. The method according to claim 4, whereinsaid determination in step (b) and optionally of step (e) is effected byemploying an antibody or a fragment thereof that specifically binds tothe Pax4 protein and optionally by employing a second antibody or afragment thereof which specifically binds to the Pax6 protein.
 12. Themethod according to claim 2 comprising(d) determining the level orstatus of Pax6 mRNA in pancreatic cells of said mammal; and/or (e)determining the level or status of Pax6 protein in pancreatic cells ofsaid mammal; and (f) comparing said level or status of Pax6 mRNA and/orPax6 protein with the corresponding level in normal pancreaticcells;wherein the term "level" denotes the amount of mRNA or proteinproduced; and, the term "status" includes that the Pax6 gene, mRNA,protein or a transcription control element, including apromoter/enhancer sequence, may bear a mutation, deletion or any othermodifications which would affect the overall activity of the gene whencompared to the wild-type normal gene product, includingpost-translational modifications of the protein, and from the comparingdetermining an absence of Pax6 mRNA or protein and thus risk ofdeveloping juvenile diabetes.
 13. The method of claim 2 wherein saidmammal is(i) an embryo; (ii) a newborn; or (iii) an adult.
 14. Themethod according to claim 2 wherein said mammal is a mouse.
 15. Themethod according to claim 2 wherein said mammal is a human.
 16. Themethod according to claim 12, wherein steps (a) and/or (b) andoptionally (d) and/or (e) are carried out in vivo.
 17. The methodaccording to claims 12, wherein steps (a) and/or (b) and optionally (d)and/or (e) are carried out in vitro.
 18. The method according to claim12 wherein said determination in step (a) and optionally in step (d) iseffected by employing(i) a nucleic acid sequence corresponding to atleast a part of the Pax4 gene encoding at least part of the Pax4 proteinand optionally a second nucleic acid sequence corresponding to at leasta part of the Pax6 gene encoding at least part of the Pax6 protein; (ii)a nucleic acid sequence complementary to the nucleic acid sequence(s) of(i); or (iii) a primer or a primer pair hybridizing to the nucleic acidsequence(s) of (i) or (ii).
 19. The method according to claim 12,wherein said determination in step (b) and optionally of step (e) iseffected by employing an antibody or a fragment thereof thatspecifically binds to the Pax4 protein and optionally by employing asecond antibody or a fragment thereof which specifically binds to thePax6 protein.
 20. The method according to claim 3 further comprising(d)determining the level or status of Pax6 mRNA in pancreatic cells of saidmammal; and/or (e) determining the level or status of Pax6 protein inpancreatic cells of said mammal; and (f) comparing said level or statusof Pax6 mRNA and/or Pax6 protein with the corresponding level in normalpancreatic cells; wherein the term "level" denotes the amount of mRNA orprotein produced; and, the term "status" includes that the Pax6 gene,mRNA, protein or a transcription control element, including apromoter/enhancer sequence, may bear a mutation, deletion or any othermodifications which would affect the overall activity of the gene whencompared to the wild-type normal gene product, includingpost-tanslational modifications of the protein, and from the comparing,determining an absence of Pax6 mRNA or protein and thus efficacy of themedicament or the gene therapy.
 21. The method of claim 3 wherein saidmammal is(i) an embryo; (ii) a newborn; or (iii) an adult.
 22. Themethod according to claim 3 wherein said mammal is a mouse.
 23. Themethod according to claim 3 wherein said mammal is a human.
 24. Themethod according to claim 20, wherein steps (a) and/or (b) andoptionally (d) and/or (e) are carried out in vivo.
 25. The methodaccording to claims 20, wherein steps (a) and/or (b) and optionally (d)and/or (e) are carried out in vitro.
 26. The method according to claim20 wherein said determination in step (a) and optionally in step (d) iseffected by employing(i) a nucleic acid sequence corresponding to atleast a part of the Pax4 gene encoding at least part of the Pax4 proteinand optionally a second nucleic acid sequence corresponding to at leasta part of the Pax6 gene encoding at least part of the Pax6 protein; (ii)a nucleic acid sequence complementary to the nucleic acid sequence(s) of(i); or (iii) a primer or a primer pair hybridizing to the nucleic acidsequence(s) of (i) or (ii).
 27. The method according to claim 20,wherein said determination in step (b) and optionally of step (e) iseffected by employing an antibody or a fragment thereof thatspecifically binds to the Pax4 protein and optionally by employing asecond antibody or a fragment thereof which specifically binds to thePax6 protein.
 28. The method according to claim 2 wherein the medicamentor treatment comprises: a compound which lowers blood glucose levels.29. The method according to claim 2 wherein the medicament or treatmentcomprises: a compound which increases cellular uptake of glucose and itsconversion to glycogen.
 30. The method of claim 2 wherein in themedicament or treatment comprises: insulin.
 31. The method of claim 29,wherein the mammal is a human, rat or mouse.
 32. The method of claim 28,wherein the mammal is a human, rat or mouse.
 33. The method of claim 30,wherein the mammal is a human, rat or mouse.
 34. The method according toclaim 32, wherein said β-cells are from an embryo or a newborn mammal.35. The method according to claim 2, wherein said β-cells are from anembryo or a newborn mammal.
 36. The method according to claim 3, whereinsaid β-cells are from an embryo or a newborn mammal.
 37. The methodaccording to claim 3, wherein said medicament affects the expressionlevel of the Pax4 gene and optionally also affects the expression of thePax6 gene at the mRNA or the protein level.
 38. The method according toclaim 3, wherein said testing is in Pax4 knockout mice that areoptionally also Pax6 knockout mice.
 39. The method according to claim 3,further comprising introducing into pancreatic or pancreatic ductalepithelial cells a Pax4 gene which is expressed and optionally furthercomprising introducing into pancreatic or pancreatic ductal epithelialcells a Pax6 gene which is expressed.
 40. A functional Pax4 protein. 41.A composition comprising the functional Pax4 protein of claim 40 andoptionally a functional Pax6 protein.